and Direct Antiglobulin (Coombs) Testing and the Crossmatch
J. Triulzi, M.D.
antiglobulin test was first introduced into clinical medicine in 1945 by
R.R. Coombs who showed that it could be used to detect non-agglutinating red
cell antibodies (indirect antiglobulin test, IAT) or sensitized red cells
(direct antiglobulin test, DAT). Most
non-agglutinating (incomplete) antibodies are IgG, although some antibodies
are IgM. These antibodies do not spontaneously cause agglutination due to a
strong electronegative charge on the red cell surface that prevents the
cells from coming into close proximity.
The antiglobulin reagent is able to bridge these negative forces.
Current antiglobulin reagent (Coombs reagent) preparations contain a
"cocktail" of monoclonal antibodies directed against human IgG and
C3. The latter is more
effective than an anti-IgM reagent for detecting IgM antibodies because a
single IgM molecule will bind numerous complement molecules to the red cell
surface and IgM antibodies tend to spontaneously dissociate from the red
Direct Antiglobulin Test (DAT)
The DAT is
used to detect IgG or C3 bound to the surface of the red cell.
In patients with hemolysis, the DAT is useful in determining whether
there is an immune etiology. Non-immune
causes of hemolysis such as DIC, thrombotic thrombocytopenic purpura,
mechanical hemolysis such as those due to artificial valves or burns,
hemoglobinopathies (sickle cell, thalassemia), red cell enzyme deficiencies
(G6PDP, pyruvate kinase), and red cell membrane defects (hereditary
spherocytosis, PNH) will have a negative DAT. Immune causes of hemolysis
including autoimmune hemolytic anemias, drug induced hemolysis, and delayed
or acute hemolytic transfusion reactions are characterized by a positive
DAT. A positive DAT can occur without hemolysis. A small proportion of
normal individuals have a
positive DAT without evidence of decreased red cell survival. Thus, a positive DAT, by itself, does not mean that the
patient has an immune hemolytic anemia.
The DAT is a
5-10 minute procedure performed by incubating patient red cells with the
antiglobulin (Coombs) reagent. A
positive DAT due to IgG is seen most frequently in patients with warm
1/3 of these patients also have C3 on the red cell membrane. IgG coated red cells may also be seen in patients who have
received an incompatible transfusion. Thus,
the DAT is routinely performed as part of all transfusion reaction
investigations. IgG bound to
the red cell surface can be eluted and its specificity determined.
Eluted autoantibodies usually bind to all red cells
(panagglutinin) but occasionally have specificity within the Rh system.
Eluted alloantibodies can usually be given a distinct
specificity.(eg. anti-D, anti-K) Red
cells sensitized with IgG may be destroyed by extravascular hemolysis.
The primary site of removal is the spleen via Fc receptors on
phagocytic cells. Factors that
determine whether hemolysis will occur include: antibody titer, number of
IgG molecules on the red cell, number of antigen sites on the red cell, IgG
subclass, and splenic function.
DAT due to complement (C3) alone is seen in patients with cold
autoantibodies, paroxysmal cold hemoglobinuria, and in some drug induced
hemolytic anemias. The
offending antibodies are typically of the IgM isotypes that efficiently bind
complement. IgM antibodies are not directly detected by the DAT, but are
detected indirectly by the presence of C3 on the red cell surface.
Cold autoimmune hemolytic anemias may be associated with
intravascular hemolysis due to complement mediated lysis.
Extravascular removal of C3 coated cells can also occur via
complement receptors on phagocytes in the liver.
patients with immune hemolysis may have a negative DAT.
Many of these patients have IgG, IgM, or IgA antibodies detectable on
the red cell only with more sensitive techniques. (MicroCoomb’s test)
Indirect Antiglobulin Test (IAT)
The IAT is
used to detect red cell antibodies in patient serum.
In clinical practice this is referred to as the "antibody
screen" and is part of the type and screen procedure.
Approximately 5% of patients have a positive IAT due to IgG
antibodies, IgM antibodies, or both. Most clinically significant alloantibodies are IgG antibodies
that react best at 37°C and are formed as a result
of previous exposure via transfusion or pregnancy.
Examples include antibodies to Rh, Kell, Kidd, and Duffy red cell
antigens. IgM antibodies are
usually not clinically significant (except for ABO antibodies) but are a
source of in-vitro serologic difficulty that may delay transfusion.
Examples include antibodies to the Lewis, I, P, M, and N red cell
antigens. IgM antibodies react
best at cold temperatures (4°C)
and are usually naturally occurring in that they do not require a
The IAT (antibody
screen) is performed by incubating patient serum with reagent screening red
cells for approximately 20 minutes and then observing for agglutination.
If the antibody screen is positive, additional testing is required to
determine the specificity of the antibody.
transfusion is necessary, patients with clinically significant red cell
alloantibodies identified in the antibody screen should receive antigen
negative red cells. Compatible
blood may be difficult to find if antigen negative blood is rare or if
multiple antibodies are present. Consultation
with the transfusion service is helpful in developing a transfusion strategy
in these cases.
crossmatch (CM) represents a special form of the IAT in which the red cells
used for testing are from the unit intended for transfusion. The purpose is
to establish in vitro compatibility in the expectation that the transfused
cells will exhibit normal in-vivo survival.
Historically, a major CM involving patient serum and donor red cells
was performed on every unit intended for transfusion. The major crossmatch
takes 20-30 minutes and is now reserved only for patients with clinically
significant red cell antibodies. The minor CM involving patient red cells
and donor plasma has not been performed for more than 20 years because
packed red cells have <70 ml of plasma. More recently it has been
recognized that patients with a negative antibody screen and no history of
red cell antibodies do not require a complete 20-30 minute crossmatch.
The chances of a
clinically significant red cell antibody being missed in a patient with a
negative antibody screen (false negative) is 1-4/10,000. Approximately 95%
of transfusions occur in patients with a negative antibody screen.
Such patients can undergo abbreviated CM testing in which only ABO
compatibility of the unit need be established.
There are two methods for abbreviated CM testing.
The “immediate spin” crossmatch (ISCM) requires only a 5 minute
incubation at room temperature with patient serum and donor red cells. For
the last 4 years the American Association of Blood Banks has also allowed an
abbreviated CM to be performed by computer confirmation of ABO compatibility
without any serologic testing: ”computer crossmatch”.
The patient must have two determinations of their ABO group on record
and a negative antibody screen. Using a validated computer system the bar
code on unit of blood can be wanded and the computer will compare the ABO of
the unit to that of the patient and indicate whether it is compatible.
Advantages of the computer crossmatch includes: faster turn-around,
computer prevents release of ABO incompatible units, lower reagent costs,
and improved quality control.
The IAT and
DAT are used to detect red cell antibodies in the serum and on the red cell,
respectively. The DAT is used
to determine whether patients with hemolysis have an immune etiology.
The IAT is used to identify clinically significant red cell
alloantibodies that are important in choosing compatible blood products.
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